The interaction of proteins with surfaces is a widely recognized phenomenon of both physiological and technological significance. An important example is the adsorption of protein allergens to the adjuvant aluminum hydroxide in allergy vaccines. An adjuvant is a compound that acts by enhancing the immune response upon vaccination. The adjuvant effect of aluminium hydroxide has been heavily investigated and numerous theories regarding the mechanism have been proposed.
Allergy vaccines for e.g. subcutaneous injection may be prepared by mixing an aqueous solution of an allergen and a solid phase carrier, e.g. aluminium hydroxide gel, to produce a mixture, wherein at least a part of the allergen is adsorbed to the solid phase and part of or none of the allergen is in the liquid phase. The solid phase carrier may serve as an adjuvant, i.e. it potentiates the immune response of the allergen, although the mechanism of the potentiation is not always fully understood. Also, the mechanism and nature of the adsorption of the allergen to the solid phase carrier is not always fully understood and may depend strongly on the type of allergen involved. Theoretically, however, the adsorption to aluminium hydroxide gels partly involves electrostatic forces. For proteins, it is believed that the phosphate groups of phosphorylated proteins also interact with the aluminium hydroxide gel and possibly to some extent replaces the hydroxide groups in the gel structure.
The protein adsorption capacity of aluminium hydroxide has been studied intensively with the model proteins ovalbumin (OA) and bovine serum albumin (BSA). Recently, studies concerning the structural impact of adsorption of protein to aluminium hydroxide have been carried out. Emission fluorescence measurements together with differential scanning calorimetry indicate that major structural alterations occur upon adsorption of OA and BSA to aluminium hydroxide (Jones et al., Effects of Adsorption to Aluminium Salt Adjuvants on the Structure and Stability of Model Protein Antigens, The Journal of Biological Chemistry, Vol, 280, pp. 13406-13414, 2005). Another study on the contrary indicates that the presence of aluminium hydroxide in an ELISA experiment helped maintaining OA in the native conformation (Houen et al., A Non-denaturing Enzyme Linked Immunosorbent Assay With Protein Preadsorbed Onto Aluminium Hydroxide, Journal of Immunological Methods, Vol. 200, pp. 99-105, 1997). OA adsorbed to aluminium hydroxide before transfer to the plastic surface of the well of a micro titer plate, maintained its ability to bind monoclonal antibodies raised towards the native form of OA. On the contrary OA not pre-incubated with aluminium hydroxide bound to monoclonal antibodies raised against heat denatured albumin. However, these techniques do not give any information on individual proteins present in a mixture of proteins.
The effect of aluminium hydroxide on structure and stability of allergens is important from several perspectives. Conformational epitopes may be lost during adsorption and immunogenicity may be altered as a consequence of storage over longer period of time.
The degree of adsorption varies with the nature of the specific allergen in question. In the case of an allergen in the form of an extract of a biological material, e.g. an extract of grass pollen allergens, the extract contains a number of different ions and molecules, which potentially interferes with the bonding of the allergens to the solid phase carrier.
The house dust mite (HDM) Dermatophagoides pteronyssinus is a major source of inhaled allergens. The protein allergens Der p 1 and Der p 2 are considered to be the two most potent allergens of the Der p allergens. The structure and enzymatic activity of Der p 1 has been well characterized. Several in vitro studies suggest that the cysteine protease activity of Der p 1 enhances the potency of the allergen e.g. by cleaving tight junction proteins in the lung epithelial and cleaving CD23 (low affinity IgE receptor) on human B-cells (Jacquet et al., Biochemical and Immunological Characterization of a Recombinant Precursor form of the House Dust Mite Allergen Der p 1 produced by Drosophila cells, Clinical and Experimental Allergy, Vol. 30, pp. 784-793, 2000). HDM vaccines based on aluminium hydroxide adjuvant contain purified HDM extract as an active pharmaceutical ingredient (API).
The allergenic activity and the potential for inducing allergic reactions may be tested for example by intradermal injection in sensitised animals, and by measurement of the change of various symptoms (Kildsgaard et al., Assessment of the in vivo allergenic potency of new allergy vaccines by intradermal testing in sensitised mice, Clinical Immunology and Allergy in Medicine, Proceedings of the 21st EAACI Congress 2002, Naples, Italy). However, such in vivo methods are laborious and time-consuming, and they necessitate the use of test animals, which is undesirable.
Up to now it has been common practise to evaluate the immunological activity of a vaccine in vitro on the basis of a measurement of the immunological activity of the solution of allergen used for the preparation of the ready-to-use solid phase carrier vaccine.
WO2005/022157 discloses an in vitro method of evaluating the immunological activity of a vaccine preparation in the form of a mixture of a molecular antigen and a carrier, wherein the mixture comprises a liquid phase and a solid phase, to which at least a part of the antigen is attached, the method comprising the steps of i) subjecting the vaccine to a measurement of the immunological activity selected from the group consisting of a) antibody binding capacity using an immunoassay employing an antigen-specific antibody bound to an antibody solid phase, b) ability to activate effector cells and c) potential for inducing anaphylaxis; and ii) using the measurement results to evaluate the immunological activity of the vaccine.
The nature of allergen adsorption to oxygen-containing metal salt adjuvants is very complex and largely unknown, and is also expected to vary among different allergens and different oxygen-containing metal salts. The object of the present invention is to provide a new in vitro method of evaluating and quantifying the immunological activity of allergy vaccine preparations based on oxygen-containing metal salt adjuvants, such as ready-to-use vaccines.